Question

How to get cells of plastic dish in staining (flow cytometry)? and how to stain the inside of the cell?

Answer 1

If the cells under investigation are adherent to a substrate pr plastic, such as fibroblasts, and cannot be shaken, one can simply dissolve JC-1 at the final concentration in complete medium and then cover the cell layer with such a solution. After 10-15 min of incubation, rinse the plate with PBS (Phosphate Buffered Saline) or other adequate saline solution two to four times, each time leaving the saline solution for 5 min in order to equilibrate, and subsequently dilute, the fluorescent probe. This allows the analysis of JC-1 fluorescence in morphologically and metabolically intact, adherent cells with microscopy techniques. If flow cytometry analysis is required, first detach cells from the substrate as gently as possible and then stain them.

After staining with JC-1, cells with high display green oragnge fluorescence owning to J aggregates. On the other hand, in cells with low JC-1 maintains its monomeric form and thus shows only green fluorescence. Because JC-1 cam also bind to membranes other than mitochondrial ones.